Competition between electrostatic interactions and halogen bonding in the protein–ligand system: structural and thermodynamic studies of 5,6-dibromobenzotriazole-hCK2α complexes

CK2 is a member of the CMGC group of eukaryotic protein kinases and a cancer drug target. It can be efficiently inhibited by halogenated benzotriazoles and benzimidazoles. Depending on the scaffold, substitution pattern, and pH, these compounds are either neutral or anionic. Their binding poses are dictated by a hydrophobic effect (desolvation) and a tug of war between a salt bridge/hydrogen bond (to K68) and halogen bonding (to E114 and V116 backbone oxygens). Here, we test the idea that binding poses might be controllable by pH for ligands with near-neutral pKa, using the conditionally anionic 5,6-DBBt and constitutively anionic TBBt as our models. We characterize the binding by low-volume Differential Scanning Fluorimetry (nanoDSF), Isothermal Calorimetry (ITC), Hydrogen/Deuterium eXchange (HDX), and X-ray crystallography (MX). The data indicate that the ligand pose away from the hinge dominates for the entire tested pH range (5.5–8.5). The insensitivity of the binding mode to pH is attributed to the perturbation of ligand pKa upon binding that keeps it anionic in the ligand binding pocket at all tested pH values. However, a minor population of the ligand, detectable only by HDX, shifts towards the hinge in acidic conditions. Our findings demonstrate that electrostatic (ionic) interactions predominate over halogen bonding.


Fig. S5
Suppl. Fig. 5. The binding mode of 4,5,6,7-TBBt in complex with hCK2α at two different pH values. The anomalous difference Fourier maps were calculated for the models without the ligands, contoured at 4.5 rmsd and shown in yellow (top panel). The composite omit electron density maps are contoured at 1 rmsd and shown in gray, the difference density map is contoured at 3 rmsd (green) and -3 rmsd (red) (bottom panel). The structure obtained at pH 7.5 has been published before (PDB 6TLL) 2 .

Fig. S6
Suppl. Fig. 6. The secondary ligand binding site in complexes of hCK2α with 5,6-DBBt (top) and TBBt (bottom) at different pH values. The anomalous difference Fourier maps were calculated for the models without the ligands, contoured at 2.5 rmsd and shown in yellow.
The structures obtained at pH 7.5 have been published before (PDB 6TLP and 6TLL) 2 .
The replicas indicate that the occupation of the secondary binding site is inconsistent in the crystals.
Only the first presented structures at each pH value (marked with ticks) were deposited in the PDB, the replicas were not refined further. The position of the second ligand molecule in the crystals clashed with the protein in the region of residue 36 and thus the ligands were in most cases modelled at half occupancy only (to match one of the alternative conformations of the protein).

Fig. S7
Suppl. Fig. 7. Results of 10 sec hydrogen deuterium exchange determined for hCK2α at pH 6.7 (upper panel) and 8.7 (lower panel) in the free form (red) and in complex with 5,6-DBBt (blue). Peptides for which the theoretical deuteration was calculated are denoted in gray.